Applying COI barcode for typing Leishmania parasites, a taxonomically challenging group. Barcode for Leishmania parasites.

The genus Leishmania was first described in 1903, and so far varying numbers of species were defined. Although recent hierarchical taxonomic schemes have increasingly used intrinsic characters to assign Leishmania organisms into different species, they are still heirs of the first classifications based primarily on extrinsic characters. Many species were first described based on ecological, epidemiological and biological characteristics. The current classification system, which proposes up to 35 species, is based on multilocus enzyme electrophoresis (MLEE), which is still the gold standard technique for the identification of Leishmania species. Although many genes have been target for Leishmania taxonomy, nearly all failures of species identification through DNA sequence analysis could be attributed to imperfect taxonomy (overlumping or overspliting) or incomplete lineage sorting, indicating that comprehensive taxonomic revision (species boundary delimitation) is crucial in the DNA studies. Many genes were already analyzed, but multilocus analyses were restricted, as far as we know, to specific groups (species or complex of species or subgenus). In a broad sense, single locus analysis has been referred as barcoding, although “traditional” barcode studies employ the Cytochrome oxidase I mitochondrial gene - COI. In all barcode studies (broad sense), Leishmania species identification considers MLEE results and none study show a total agreement between DNA sequence analysis and MLEE, and some disagreements were also detected among DNA analysis. Our idea is to evaluate the potential of COI as a barcode for Leishmania species identification. Up to now, we already analysed more than 200 strains, representing different species. Comparing with other markers, the results obtained so far indicate the best overlap with the actual classification. A total of 487 sites were analysed and several group or species-specific sites were detected. COI allowed the identification of all L. (Viannia) species and most of L. (Leishmania), point to a revision of L. mexicana complex, as L. venezuelensis was not validated and some non-typical L. amazonensis (Bolivian strains) were placed closer to L. mexicana than to L. amazonensis. Some authors argue that hsp70 gene is a good marker and could overcome the limitations of MLEE, besides promote a taxonomic revision. However, this gene cannot distinguish some species. Our group have analyzed hsp70 gene of many Leishmania strains representing Brazilian species and in some cases the species boundary delimitation was not evident. There are some reports showing hsp70 pseudogene in Leishmania. Pseudogene formation after duplication has been recognized as a relevant event responsible for species-specific differences in gene content among Leishmania. But, undoubtedly, sampling is responsible for the main difficulty to get a consensus among Leishmania’ taxonomic studies and finally minimally define species boundary delimitation. The studies are limited to few samples of each species and most of the time limited to reference strains. Increasing the difficulty, different studies employ different strains, limiting the comparison of results. Recombination disrupts signals of differentiation in nuclear genome, and since mitochondrial DNA is non-recombining it can be informative for taxonomic purpose. We encourage the use of COI barcode for Leishmania, a taxonomically challenging group.